Anti-TIE2 antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to Tie2 and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human Tie2 and block the interaction between Tie2 and one or more Tie2 ligands such as angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), angiopoietin 3 (Ang3) and/or angiopoietin 4 (Ang4). The antibodies of the invention are useful, inter alia, for the treatment of diseases and disorders associated with one or more Tie2 biological activities including angiogenesis. In certain embodiments, pairs of activating Tie-2 antibodies showed an additive effect on the treatment of influenza infection when combined with anti-influenza HA. In other embodiments, prophylactic administration pairs of activating Tie2 antibodies delayed death and improved survival in a lethal model of E. coli intoxication (sepsis) over isotype/untreated controls.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.15/373,903, filed on Dec. 9, 2016, now U.S. Pat. No. 10,023,641, whichis a continuation of U.S. application Ser. No. 14/671,494, filed on Mar.27, 2015, now U.S. Pat. No. 9,546,218, which is a divisional of U.S.application Ser. No. 13/586,911, filed on Aug. 16, 2012, now U.S. Pat.No. 9,017,670, which claims the benefit under 35 U.S.C. § 119(e) ofprovisional application Nos. 61/525,308 filed on Aug. 19, 2011,61/587,213 filed on Jan. 17, 2012, and 61/674,405, filed Jul. 23, 2012,the disclosures of which are herein incorporated by reference in theirentireties.

SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“493878-Sequence.txt”, created on Feb. 28, 2017 and containing 111,030bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for human Tie2.

BACKGROUND

Angiogenesis is the biological process whereby new blood vessels areformed. Aberrant angiogenesis is associated with several diseaseconditions including, e.g., proliferative retinopathies, rheumatoidarthritis and psoriasis. In addition, it is well established thatangiogenesis is critical for tumor growth and maintenance. Tie2 is asingle transmembrane tyrosine kinase receptor that has been localized tothe endothelial cells of forming blood vessels and has been shown toplay a role in angiogenesis. Tie2 ligands include the angiopoietins(e.g., Ang1, Ang2, Ang3 and Ang4). Blocking the interaction between Tie2and one or more of its ligands is expected to have beneficialtherapeutic effects in settings where it is advantageous to limit orblock angiogenesis.

Antibodies to Tie2 are mentioned, e.g., in U.S. Pat. Nos. 6,365,154 and6,376,653. Nonetheless, there remains a need in the art for novelmolecules capable of binding to Tie2, especially anti-Tie2 antibodiesthat can block the interaction of Tie2 with one or more Tie2 ligandssuch as Ang2. Such molecules would be useful for various therapeutic anddiagnostic purposes.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that bind human Tie2. Theantibodies of the invention are useful, inter alia, for inhibitingTie2-mediated signaling and for treating diseases and disorders causedby or related to Tie2 activity and/or signaling. According to certainembodiments, the antibodies of the invention block the interactionbetween Tie2 and one or more Tie2 ligands such as Ang1, Ang2, Ang3,and/or Ang4.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

The present invention provides anti-Tie2 antibodies that havesubstantially the same binding characteristics as any of the exemplaryanti-Tie2 antibodies described herein. The present invention includescell lines that produce the anti-Tie2 antibodies described herein. Asnon-limiting examples, cell lines which produce the exemplary antibodiesH1M2055N and H2aM2760N were deposited under terms in accordance with theBudapest Treaty with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 on Dec. 2, 2011. Thedeposited cell lines have been assigned the following accession numbers:PTA-12295 (H1M2055N) and PTA-12296 (H2aM2760N).

Exemplary anti-Tie2 antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs), light chainvariable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-Tie2antibodies. Table 2 sets forth the nucleic acid sequence identifiers ofthe HCVRs, LCVRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of theexemplary anti-Tie2 antibodies.

The present invention provides antibodies or antigen-binding fragmentsthereof that specifically bind Tie2, comprising an HCVR comprising anamino acid sequence selected from any of the HCVR amino acid sequenceslisted in Table 1, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising an HCVR and anLCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVRamino acid sequences listed in Table 1 paired with any of the LCVR aminoacid sequences listed in Table 1. According to certain embodiments, thepresent invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR/LCVR amino acid sequence pair containedwithin any of the exemplary anti-Tie2 antibodies listed in Table 1. Incertain embodiments, the HCVR/LCVR amino acid sequence pair is selectedfrom the group consisting of SEQ ID NOs: 20/28, 36/44 and 52/60.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a heavy chainCDR1 (HCDR1) comprising an amino acid sequence selected from any of theHCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a heavy chainCDR2 (HCDR2) comprising an amino acid sequence selected from any of theHCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a heavy chainCDR3 (HCDR3) comprising an amino acid sequence selected from any of theHCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a light chainCDR1 (LCDR1) comprising an amino acid sequence selected from any of theLCDR1 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a light chainCDR2 (LCDR2) comprising an amino acid sequence selected from any of theLCDR2 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a light chainCDR3 (LCDR3) comprising an amino acid sequence selected from any of theLCDR3 amino acid sequences listed in Table 1 or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising an HCDR3 andan LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of theHCDR3 amino acid sequences listed in Table 1 paired with any of theLCDR3 amino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acidsequence pair contained within any of the exemplary anti-Tie2 antibodieslisted in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acidsequence pair is selected from the group consisting of SEQ ID NOs:24/34, 42/50 and 58/66.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind Tie2, comprising a set of sixCDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) containedwithin any of the exemplary anti-Tie2 antibodies listed in Table 1. Incertain embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3amino acid sequences set is selected from the group consisting of SEQ IDNOs: 22, 24, 26, 30, 32, 34; 38, 40, 42, 46, 48, 50; and 54, 56, 58, 62,64, 66.

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof that specifically bind Tie2,comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3) contained within an HCVR/LCVR amino acid sequence pair asdefined by any of the exemplary anti-Tie2 antibodies listed in Table 1.For example, the present invention includes antibodies orantigen-binding fragments thereof that specifically bind Tie2,comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acidsequences set contained within an HCVR/LCVR amino acid sequence pairselected from the group consisting of SEQ ID NOs: 20/28, 36/44 and52/60. Methods and techniques for identifying CDRs within HCVR and LCVRamino acid sequences are well known in the art and can be used toidentify CDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-Tie2 antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention includes antibodies and antigen-binding fragmentsthereof comprising the heavy and light chain CDR amino acid sequences(HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) found within any of theexemplary anti-Tie2 antibodies described herein, including theantibodies produced by the deposited cell lines PTA-12295 and PTA-12296.The present invention also includes antibodies and antigen-bindingfragments thereof comprising the heavy and light chain variable domainamino acid sequences (HCVR and LCVR) found within any of the exemplaryanti-Tie2 antibodies described herein, including the antibodies producedby the deposited cell lines PTA-12295 and PTA-12296.

The present invention includes any of the exemplary anti-Tie2 antibodiesdescribed herein having a modified glycosylation pattern. In someapplications, modification to remove undesirable glycosylation sites maybe useful, or an antibody lacking a fucose moiety present on theoligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, modification of galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides pharmaceutical compositionscomprising an anti-Tie2 antibody as described herein and apharmaceutically acceptable carrier. In a related aspect, the inventionfeatures compositions which comprise a combination of an anti-Tie2antibody and a second therapeutic agent. Exemplary agents that may beadvantageously combined with an anti-Tie2 antibody include, withoutlimitation, other agents that inhibit anti-Tie2 activity (includingother antibodies or antigen-binding fragments thereof, peptideinhibitors, small molecule antagonists, etc.) and/or agents whichinterfere with Tie2 upstream or downstream signaling.

In yet another aspect, the invention provides methods for inhibitingTie2 activity using an anti-Tie2 antibody or antigen-binding portion ofan antibody of the invention, wherein the therapeutic methods compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment of anantibody of the invention. The disorder treated is any disease orcondition which is improved, ameliorated, inhibited or prevented byremoval, inhibition or reduction of Tie2 activity. The anti-Tie2antibody or antibody fragment of the invention may function to block theinteraction between Tie2 and a Tie2 binding partner (e.g., Ang1, Ang2,Ang3, and/or Ang4), or otherwise inhibit the signaling activity of Tie2.

The present invention also includes the use of an anti-Tie2 antibody orantigen binding portion of an antibody of the invention in themanufacture of a medicament for the treatment of a disease or disorderrelated to or caused by Tie2 activity in a patient.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Linear depiction of full-length human Tie2 and various deletionconstructs used to map the epitope of the anti-Tie2 antibodies of thepresent invention. Numbers above the constructs indicate the amino acidboundaries of the constructs relative to the full-length Tie2 molecule(SEQ ID NO:1).

FIG. 2: Histogram showing the extent of angiopoietin (hAng1, hAng2,mAng3 or hAng4) binding to a hTie2-coated sensor tip pre-treated withanti-Tie2 antibody H4H2055N or Control I.

FIG. 3: Histogram showing the extent of anti-Tie2 antibody (H4H2055N orControl I) binding to a hTie2-coated sensor tip pre-treated with 100 nMof angiopoietin (hAng1, hAng2, mAng3 or hAng4).

FIGS. 4A and 4B: FIG. 4A shows the H29 tumor growth curves in micefollowing administration of anti-Tie2 antibody H2M2055N (▪) or Fccontrol (●). Downward arrow indicates the treatment start date. FIG. 4Bshows the tumor growth from the start of treatment in mice treated withanti-Tie2 antibody H2M2055N or Fc control. Asterisk (*) indicates p<0.05Mann Whitney non-parametric two-tailed t-test.

FIG. 5: H29 tumor vessel density measured at the end of the experimentin mice treated with anti-Tie2 antibody H2M2055N or Fc control. Asterisk(*) indicates p<0.05 Mann Whitney non-parametric two-tailed t-test.

FIGS. 6A and 6B: FIG. 6A shows the Colo305 tumor growth curves in micefollowing administration of anti-Tie2 antibody H2M2055N (▪) or Fccontrol (●). Downward arrow indicates the treatment start date. FIG. 6Bshows the tumor growth from the start of treatment in mice treated withanti-Tie2 antibody H2M2055N or Fc control. Asterisk (*) indicates p<0.05Mann Whitney non-parametric two-tailed t-test.

FIG. 7: Colo205 tumor vessel density measured at the end of theexperiment in mice treated with anti-Tie2 antibody H2M2055N or Fccontrol. Asterisk (*) indicates p<0.05 Mann Whitney non-parametrictwo-tailed t-test.

FIG. 8: Shows the percent permeability of anti-Tie2 antibody pairsH4H2332P/H4H2339P and H4H2340P/H4H2339P in comparison with TNF-α orhIgG4 controls in an in vitro transwell permeability assay.

FIG. 9: Shows therapeutic administration of activating Tie2 mAbs incombination with anti-influenza A hemagglutinin mAb improves survivalcompared to treatment with the anti-hemagglutinin specific mAb aloneafter lethal challenge with Influenza A.

FIG. 10: Shows that prophylactic administration of activating Tie2 mAbs,H4H2340P/H4H2339P delays death and improves survival in an E. coli0111:B4 LPS Sepsis model over isotype/untreated controls.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expressions “Tie2” and “Tie2 fragment,” as used herein refer to thehuman Tie2 protein or fragment unless specified as being from anon-human species (e.g., “mouse Tie2,” “mouse Tie2 fragment,” “monkeyTie2,” “monkey Tie2 fragment,” etc.). A “Tie2 fragment” is any portionof Tie2 having fewer amino acids than the full-length Tie2 molecule andwhich is capable of binding to a Tie2 ligand. Human Tie2 has the aminoacid sequence set forth in SEQ ID NO:1. Amino acid sequences of Tie2molecules from non-human species (e.g., mouse, monkey, rabbit, dog, pig,etc.) are available from public sources such as GenBank (e.g., GenBankaccession numbers NP_038718.2 (mouse); NP_001099207.1 (rat); etc).

The term “Tie2 ligand,” as used herein, means a protein with which theTie2 protein interacts to transmit a biological signal in vivo. The term“Tie2 ligand” includes any of the angiopoietins, including, e.g., Ang1,Ang2, Ang3 and/or Ang4. The term “Ang1,” as used herein, means a proteincomprising the amino acid sequence of SEQ ID NO:15, or a portion thereofwhich is capable of interacting with Tie2. The term “Ang2,” as usedherein, means a protein comprising the amino acid sequence of SEQ IDNO:16, or a portion thereof which is capable of interacting with Tie2.The term “Ang3,” as used herein, means a protein comprising the aminoacid sequence of SEQ ID NO:17, or a portion thereof which is capable ofinteracting with Tie2. The term “Ang4,” as used herein, means a proteincomprising the amino acid sequence as set forth in of SEQ ID NO:18, or aportion thereof which is capable of interacting with Tie2.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-Tie2 antibody (or antigen-binding portion thereof) may be identicalto the human germline sequences, or may be naturally or artificiallymodified. An amino acid consensus sequence may be defined based on aside-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2 (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” human Tie2, as used in the context of the present invention,includes antibodies that bind human Tie2 or portion thereof with a K_(D)of less than about 1000 nM, less than about 500 nM, less than about 300nM, less than about 200 nM, less than about 100 nM, less than about 90nM, less than about 80 nM, less than about 70 nM, less than about 60 nM,less than about 50 nM, less than about 40 nM, less than about 30 nM,less than about 20 nM, less than about 10 nM, less than about 5 nM, lessthan about 4 nM, less than about 3 nM, less than about 2 nM, less thanabout 1 nM or less than about 0.5 nM, as measured in a surface plasmonresonance assay. (See, e.g., Example 3, herein). An isolated antibodythat specifically binds human Tie2 may, however, have cross-reactivityto other antigens, such as Tie2 molecules from other (non-human)species.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to Tie2: (i) interferes with theinteraction between Tie2 or a Tie2 fragment and a Tie2 ligand (e.g., anangiopoietin), and/or (ii) results in inhibition of at least onebiological function of Tie2. The inhibition caused by a Tie2neutralizing or blocking antibody need not be complete so long as it isdetectable using an appropriate assay. Exemplary assays for detectingTIE2 inhibition are described herein.

The anti-Tie2 antibodies disclosed herein may comprise one or more aminoacid substitutions, insertions and/or deletions in the framework and/orCDR regions of the heavy and light chain variable domains as compared tothe corresponding germline sequences from which the antibodies werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-Tie2 antibodies comprisingvariants having one or more conservative substitutions as compared withthe HCVR, LCVR and/or CDR amino acid sequences found within theexemplary anti-Tie2 antibodies disclosed herein. For example, thepresent invention includes anti-Tie2 antibodies having HCVR, LCVR,and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6or fewer, 4 or fewer, etc. conservative amino acid substitutionsrelative to any of the HCVR, LCVR, and/or CDR amino acid sequences ofthe anti-Tie2 antibodies disclosed herein.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Biological Characteristics of the Antibodies

The present invention includes antibodies that block the interactionbetween Tie2 and a Tie2 ligand. As used herein, the expression “blocksthe interaction between Tie2 and a Tie2 ligand” means that, in an assayin which the physical interaction between Tie2 and a Tie2 ligand can bedetected and/or quantified, the addition of an antibody of the inventionreduces the interaction between Tie2 and the Tie2 ligand (e.g., Ang1,Ang2, Ang3 and/or Ang4) by at least 50%. A non-limiting, exemplary assaythat can be used to determine if an antibody blocks the interactionbetween human Tie2 and a Tie2 ligand is illustrated in Example 5,herein. In one exemplary embodiment of this assay format, antibodies aremixed with Tie2 protein, and then the antibody/Tie2 mixture is appliedto a surface coated with a Tie 2 ligand (in this case, Ang2 protein).After washing away unbound molecules, the amount of Tie2 bound to theAng2-coated surface is measured. By using varying amounts of antibody inthis assay format, the amount of antibody required to block 50% of Tie2binding to Ang2 can be calculated and expressed as an IC₅₀ value. Theformat of this assay can be reversed such that Tie2 is coated tosurface, antibody is added to the Tie2-coated surface, unbound antibodyis washed away, and then a Tie2 ligand is added to the antibody-treatedTie2 surface. The present invention includes anti-Tie2 antibodies thatexhibit an IC₅₀ of less than about 100 nM when tested in a Tie2/Tie2ligand binding assay as illustrated in Example 5, or a substantiallysimilar assay. For example, the invention includes anti-Tie2 antibodiesthat exhibit an IC₅₀ of less than about 100, 90, 80, 70, 60, 50, 40, 30,20, 19, 18, 17, 16, 15, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4,0.3, or 0.2 nM when tested in a Tie2/Tie2 ligand binding assay asillustrated in Example 5, or a substantially similar assay.

Another assay format that can be used to determine whether an antibodyblocks the interaction between human Tie2 and a Tie2 ligand isillustrated in Example 6, herein. In this assay format, a cell line isused which is engineered to express human Tie2 on its surface, and whichalso includes a reporter construct that causes a detectable signal to beexpressed when Tie2 interacts with a Tie2 ligand. The engineered cellsare treated with anti-Tie2 antibodies and with Tie2 ligand and thereporter signal is measured. By using varying amounts of antibody inthis assay format, the amount of antibody required to inhibit 50% of thereporter signal observed in the absence of antibody can be calculatedand expressed as an IC₅₀ value. The present invention includes anti-Tie2antibodies that exhibit an IC₅₀ of less than about 20 nM when tested ina Tie2/Tie2 ligand binding assay as illustrated in Example 6, or asubstantially similar assay. For example, the invention includesanti-Tie2 antibodies that exhibit an IC₅₀ of less than about 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4,0.3, 0.2 or 0.1 nM when tested in a Tie2/Tie2 ligand binding assay asdescribed in Example 6, or a substantially similar assay.

The invention also includes Tie2 activating antibodies. For example, theexemplary anti-Tie2 antibodies of the invention H4H2340P, H4H2339P, andH4H2332P were shown to activate Tie2 activity in in vitro and in vivoassays. (See Examples 12 and 13, and Table 10 herein).

Epitope Mapping and Related Technologies

The human Tie2 protein contains the following domains: an Ig1 domain, anIg2 domain, an EGF repeat domain, an Ig3 domain, and a fibronectinrepeat (FN) domain (including FN1, FN2 and FN3). These domains aregraphically depicted in FIG. 1. The present invention includes anti-Tie2antibodies which bind specifically to an epitope within one or more ofthe following regions: (a) the Ig1-Ig2-EGF domains (SEQ ID NO:7); (b)the Ig2-EGF domains (SEQ ID NO:8); (c) the EGF domain (SEQ ID NO:9); (d)the Ig3-FN domains (SEQ ID NO:10); and/or (e) the FN domains (SEQ IDNO:11). (See Examples 3 and 4).

According to certain embodiments, the present invention providesanti-Tie2 antibodies which interact with one or more amino acids foundwithin the Ig1 and/or Ig2 domains of Tie2. The epitope(s) may consist ofone or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidslocated within the Ig1 and/or Ig2 domains of Tie2. Alternatively, theepitope may consist of a plurality of non-contiguous amino acids (oramino acid sequences) located within the Ig1 and/or Ig2 domains of Tie2.According to certain embodiments of the present invention, anti-Tie2antibodies are provided which interact with one or more amino acidslocated within one or more amino acid segments selected from the groupconsisting of amino acids 96-106 of SEQ ID NO:7, amino acids 139-152 ofSEQ ID NO:7; and amino acids 166-175 of SEQ ID NO:7. For example, thepresent invention includes anti-Tie2 antibodies which interact with atleast one amino acid within each of the aforementioned segments (i.e.,within each of amino acids 96-106, 139-152, and 166-175 of SEQ ID NO:7).According to certain embodiments of the present invention, antibodieswhich interact with amino acids 139-152 and/or 166-175 of SEQ ID NO:7are capable of blocking the interaction between Tie2 and one or moreTie2 ligands, such as, e.g., Ang2 (see Examples 4-6, herein).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. (See, e.g.,Example 4 herein). In general terms, the hydrogen/deuterium exchangemethod involves deuterium-labeling the protein of interest, followed bybinding the antibody to the deuterium-labeled protein. Next, theprotein/antibody complex is transferred to water to allowhydrogen-deuterium exchange to occur at all residues except for theresidues protected by the antibody (which remain deuterium-labeled).After dissociation of the antibody, the target protein is subjected toprotease cleavage and mass spectrometry analysis, thereby revealing thedeuterium-labeled residues which correspond to the specific amino acidswith which the antibody interacts. See, e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem.73:256A-265A.

The present invention includes anti-Tie2 antibodies that bind to thesame epitope as any of the specific exemplary antibodies describedherein (e.g., antibodies H1M2055N, and H2aM2760N, produced fromdeposited cell lines PTA-12295 and PTA-12296, respectively or antibodiesH4H2332P, H4H2339P, and H4H2340P). Likewise, the present invention alsoincludes anti-Tie2 antibodies that compete for binding to Tie2 or a Tie2fragment with any of the specific exemplary antibodies described herein(e.g., antibodies H1M2055N, and H2aM2760N, produced from deposited celllines PTA-12295 and PTA-12296, respectively, or antibodies H4H2332P,H4H2339P, and H4H2340P).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-Tie2 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-Tie2antibody of the invention, the reference antibody is allowed to bind toa Tie2 protein or peptide under saturating conditions. Next, the abilityof a test antibody to bind to the Tie2 molecule is assessed. If the testantibody is able to bind to Tie2 following saturation binding with thereference anti-Tie2 antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-Tie2 antibody. Onthe other hand, if the test antibody is not able to bind to the Tie2molecule following saturation binding with the reference anti-Tie2antibody, then the test antibody may bind to the same epitope as theepitope bound by the reference anti-Tie2 antibody of the invention.Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, Biacore, flow cytometryor any other quantitative or qualitative antibody-binding assayavailable in the art. In accordance with certain embodiments of thepresent invention, two antibodies bind to the same (or overlapping)epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibodyinhibits binding of the other by at least 50% but preferably 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodiesare deemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies aredeemed to have “overlapping epitopes” if only a subset of the amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

To determine if an antibody competes for binding with a referenceanti-Tie2 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a Tie2 molecule under saturating conditions followedby assessment of binding of the test antibody to the Tie2 molecule. In asecond orientation, the test antibody is allowed to bind to a Tie2molecule under saturating conditions followed by assessment of bindingof the reference antibody to the Tie2 molecule. If, in bothorientations, only the first (saturating) antibody is capable of bindingto the Tie2 molecule, then it is concluded that the test antibody andthe reference antibody compete for binding to Tie2. As will beappreciated by a person of ordinary skill in the art, an antibody thatcompetes for binding with a reference antibody may not necessarily bindto the same epitope as the reference antibody, but may sterically blockbinding of the reference antibody by binding an overlapping or adjacentepitope.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human Tie2.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to Tie2 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-Tie2 antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies but that retain the ability to bind human Tie2.Such variant antibodies and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, the anti-Tie2antibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-Tie2 antibody or antibody fragment that isessentially bioequivalent to an anti-Tie2 antibody or antibody fragmentof the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-Tie2 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-Tie2 antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-Tie2antibodies bind to human Tie2 but not to Tie2 from other species. Thepresent invention also includes anti-Tie2 antibodies that bind to humanTie2 and to Tie2 from one or more non-human species. For example, theanti-Tie2 antibodies of the invention may specifically bind to humanTie2 as well as to a rodent Tie2 (e.g., Tie2 from mouse or rat). Anexemplary construct that can be used to determine whether an antibodyspecifically binds mouse Tie2 is the construct having the amino acidsequence of SEQ ID NO:6; an exemplary construct that can be used todetermine whether an antibody specifically binds rat Tie2 is theconstruct having the amino acid sequence of SEQ ID NO:5. The use ofthese constructs to assess anti-Tie2 antibody binding is illustrated inExample herein.

Immunoconjugates

The invention encompasses anti-Tie2 monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-Tie2 antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor human Tie2 or a fragment thereof, and the other arm of theimmunoglobulin is specific for a second therapeutic target or isconjugated to a therapeutic moiety such as a trypsin inhibitor.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-Tie2 antibodies or antigen-binding fragments thereof of the presentinvention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-waterand water-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. When anantibody of the present invention is used for treating a condition ordisease associated with Tie2 activity in an adult patient, it may beadvantageous to intravenously administer the antibody of the presentinvention normally at a single dose of about 0.01 to about 20 mg/kg bodyweight, more preferably about 0.02 to about 7, about 0.03 to about 5, orabout 0.05 to about 3 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering Tie2antibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms. In various embodiments,certain antibodies and antigen-binding fragments of the invention (e.g.,antibodies comprising the CDRs or variable regions of H4H2332P, H4H2339Por H4H2340P) can be used to treat or prevent vascular leak, such as forexample, vascular leak associated with infections (e.g., influenza orsepsis), stroke, transplants, and eye diseases. In some cases, certaincombinations of these antibodies can be used to treat and/or preventsuch conditions (e.g., influenza infection and/or sepsis). For example,a combination of H4H2332P and H4H2339P, or a combination of H4H2340P andH4H2339P can be used.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with Tie2 activity, including diseases or disordersassociated with angiogenesis. The antibodies and antigen-bindingfragments of the present invention may be used to treat, e.g., primaryand/or metastatic tumors arising in the brain and meninges, oropharynx,lung and bronchial tree, gastrointestinal tract, male and femalereproductive tract, muscle, bone, skin and appendages, connectivetissue, spleen, immune system, blood forming cells and bone marrow,liver and urinary tract, and special sensory organs such as the eye. Incertain embodiments, the antibodies and antigen-binding fragments of theinvention are used to treat one or more of the following cancers: renalcell carcinoma, pancreatic carcinoma, breast cancer, prostate cancer,malignant gliomas, osteosarcoma, colorectal cancer, malignantmesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer,non-small cell lung cancer, synovial sarcoma, thyroid cancer, ormelanoma. In certain embodiments, the antibodies and antigen-bindingfragments of the invention are used to treat or prevent influenzainfection or sepsis.

Combination Therapies

The present invention includes therapeutic administration regimens whichcomprise administering an anti-Tie2 antibody of the present invention incombination with at least one additional therapeutically activecomponent. Non-limiting examples of such additional therapeuticallyactive components include, for example, another Tie2 antagonist (e.g.,an anti-Tie2 antibody), an antagonist of epidermal growth factorreceptor (EGFR) (e.g., anti-EGFR antibody [e.g., cetuximab orpanitumumab] or small molecule inhibitor of EGFR activity [e.g.,gefitinib or erlotinib]), an antagonist of another EGFR family membersuch as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 oranti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4activity), an antagonist of EGFRvIII (e.g., an antibody thatspecifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMETantibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-rafinhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-αinhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g.,an anti-PDGFR-β antibody), a VEGF antagonist (e.g., a VEGF-Trap, see,e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a“VEGF-inhibiting fusion protein”), anti-VEGF antibody (e.g.,bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g.,sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., ananti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), anAng2 antagonist (e.g., an anti-Ang2 antibody disclosed in US2011/0027286 such as H1H685P), etc. Other agents that may bebeneficially administered in combination with the anti-Tie2 antibodiesof the invention include cytokine inhibitors, including small-moleculecytokine inhibitors and antibodies that bind to cytokines such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17,IL-18, or to their respective receptors.

The present invention also includes therapeutic combinations comprisingany of the anti-Tie2 antibodies mentioned herein and an inhibitor of oneor more of VEGF, Ang2, DLL4, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R,B-raf, PDGFR-α, PDGFR-β, or any of the aforementioned cytokines, whereinthe inhibitor is an aptamer, an antisense molecule, a ribozyme, ansiRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fabfragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAbfragment; or other engineered molecules, such as diabodies, triabodies,tetrabodies, minibodies and minimal recognition units). The anti-Tie2antibodies of the invention may also be administered in combination withantivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. Theanti-Tie2 antibodies of the invention may also be administered as partof a treatment regimen that also includes radiation treatment and/orconventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan anti-Tie2 antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-Tie2 antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-Tie2 antibody ofthe present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

Diagnostic Uses of the Antibodies

The anti-Tie2 antibodies of the present invention may also be used todetect and/or measure Tie2 in a sample, e.g., for diagnostic purposes.For example, an anti-Tie2 antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofTie2. Exemplary diagnostic assays for Tie2 may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-Tie2 antibodyof the invention, wherein the anti-Tie2 antibody is labeled with adetectable label or reporter molecule. Alternatively, an unlabeledanti-Tie2 antibody can be used in diagnostic applications in combinationwith a secondary antibody which is itself detectably labeled. Thedetectable label or reporter molecule can be a radioisotope, such as ³H,¹⁴C, ³²P, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, beta-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure Tie2 ina sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in Tie2 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of Tie2 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of Tie2 in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal Tie2 levels or activity) will be measured to initiallyestablish a baseline, or standard, level of Tie2. This baseline level ofTie2 can then be compared against the levels of Tie2 measured in samplesobtained from individuals suspected of having a Tie2 related disease orcondition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Control Constructs Used in the Following Examples

An exemplary control construct (anti-Tie2 antibody) was included inseveral experiments described below for comparative purposes. Theantibody, referred to as Control I, is a chimeric anti-Tie2 antibodywith mouse heavy and light chain variable domains having the amino acidsequences of the corresponding domains of “12H8”, as set forth in U.S.Pat. No. 6,376,653. The constant domain of this antibody is human IgG4.

Example 1. Generation of Human Antibodies to Human Tie2

Several human anti-Tie2 antibodies were generated by immunizing aVELOCIMMUNE® mouse with human Tie2 antigen according to standard methods(see, e.g., U.S. Pat. No. 6,596,541). Using this technique, severalanti-Tie2 antibodies were obtained; exemplary antibodies generated inthis manner, and their corresponding biological characteristics, aredescribed in detail in the following Examples and include the antibodiesdesignated H2aM2760N, H2aM2761N, H1M2055N, H1H2304B, H1H2317B, H1H2322B,H1H2324B, H1H2331B, H1H2332B, H1H23335, H1H2337B, H1H2338B, H1H2339B,H1H2340B, and H4H2055N. The H1M, H2M, H1H, etc. prefixes on the antibodydesignations used herein indicate the particular Fc region of theantibody. For example, an “H2M” antibody has a mouse IgG2 Fc, whereas an“H4H” antibody has a human IgG4 Fc. As will be appreciated by a personof ordinary skill in the art, an Fc region of an antibody can bemodified or replaced with a different Fc region, but the variabledomains (including the CDRs) will remain the same.

Hybridomas which produce the anti-Tie2 antibodies H1M2055N, andH2aM2760N were deposited under terms in accordance with the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 on Dec. 2, 2011, underaccession numbers PTA-12295 (H1M2055N) and PTA-12296 (H2aM2760N).

Example 2: Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-Tie2 antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4H2332P 20 22 24 26 28 3032 34 H4sH2339 36 38 40 42 44 46 48 50 H4sH2340 52 54 56 58 60 62 64 66

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4H2332P 19 2123 25 27 29 31 33 H4sH2339 35 37 39 41 43 45 47 49 H4sH2340 51 53 55 5759 61 63 65

Example 3. Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Human Monoclonal Anti-Tie2 Antibodies

Binding affinities and kinetic constants of human monoclonal anti-Tie2antibodies were determined by surface plasmon resonance at 25° C. and37° C. (Tables 3-6). Measurements were conducted on a Biacore 2000 orT200 instrument.

For antibodies with a mouse constant region (designated H1M or H2M),antibodies were immobilized onto an anti-mouse Fc sensor surface anddifferent concentrations of human, mouse or rat Tie2 constructs(hTie2-His, mTie2-hFc and rTie2-hFc) were injected over the antibodycaptured surface. For antibodies in the human IgG format (designated H1Hor H4H), either an anti-human Fc sensor surface (hTie2-His andhTie2-mFc) or anti-human Fab sensor surface (mTie2-hFc and rTie2-hFc)was employed depending on the Tie-2 construct applied to the antibodycaptured surface. Amino acid sequences of the constructs used in thisExample are as follows: hTie2-His (SEQ ID NO:2); hTie2-hFc (SEQ IDNO:3); hTie2-mFc (SEQ ID NO:4); rTie2-hFc (SEQ ID NO:5); and mTie2-hFc(SEQ ID NO:6).

Kinetic rate constants—association rate (ka) and dissociation rate (kd)were determined by fitting the real time binding sensorgrams to a 1:1binding model with mass transport limitation using Scrubber 2.0 curvefitting software. The equilibrium dissociation constant (K_(D)) anddissociative half-life (T½) were calculated from the kinetic rateconstants as: K_(D) (M)=kd/ka; and T½ (min)=(In2/(60*kd).). As shown inTables 3-4, several antibodies demonstrated high affinity binding tohTie2 at both temperatures tested. In addition H1M2055N, H1H2332B,H1H2337B, H1H2340B and H4H2055N exhibited significant binding to mouseand rat Tie2 (Tables 5-6).

TABLE 3 Biacore binding affinities of human mAbs to hTie2 at 25° C.Binding at 25° Mab capture format T½ Antibody Analyte ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) (min) H2aM2760N hTie2-His 2.91E+04 1.84E−04 6.32E−09 63hTie2-hFc NT NT NT NT H2aM2761N hTie2-His 3.33E+04 1.01E−04 3.02E−09 115hTie2-hFc NT NT NT NT H1M2055N hTie2-His 5.75E+05 1.34E−04 2.33E−10 86hTie2-hFc NT NT NT NT H1H2304B hTie2-His 1.99E+05 1.32E−03 6.66E−09 9hTie2-mFc 5.80E+05 3.75E−05 6.46E−11 308 H1H2317B hTie2-His 1.43E+059.26E−04 6.50E−09 12 hTie2-mFc 3.90E+05 2.36E−05 6.10E−11 489 H1H2322BhTie2-His 1.67E+05 9.76E−04 5.84E−09 12 hTie2-mFc 4.80E+05 1.39E−052.90E−11 829 H1H2324B hTie2-His 2.40E+05 9.25E−04 3.86E−09 12 hTie2-mFc6.30E+05 5.92E−05 9.39E−11 195 H1H2331B hTie2-His 3.63E+04 1.17E−033.22E−08 10 hTie2-mFc 8.20E+04 1.24E−04 1.52E−09 93 H1H2332B hTie2-His8.89E+04 2.23E−03 2.51E−08 5 hTie2-mFc 1.25E+05 2.13E−04  1.1E−09 54H1H2333S hTie2-His 5.92E+04 3.09E−04 5.21E−09 37 hTie2-mFc NB NB NB NBH1H2337B hTie2-His 6.47E+04 6.49E−04 1.00E−08 18 hTie2-mFc 1.20E+051.11E−04 9.30E−10 104 H1H2338B hTie2-His 4.93E+04 3.44E−04 6.99E−09 34hTie2-mFc NB NB NB NB H1H2339B hTie2-His 5.58E+04 1.25E−03 2.24E−08 9hTie2-mFc 1.51E+05 1.02E−04 6.70E−10 114 H1H2340B hTie2-His 7.01E+043.45E−03 4.92E−08 3 hTie2-mFc 1.65E+05 1.91E−04 1.16E−09 60 H4H2055NhTie2-His 3.82E+05 4.84E−04 1.27E−09 24 hTie2-mFc 1.27E+06 9.92E−057.80E−11 116 Control I hTie2-His 6.53E+04 4.57E−04 7.00E−09 25 hTie2-mFc1.45E+05 1.16E−04 8.03E−10 99 NB = no binding under the conditionstested NT = not tested

TABLE 4 Biacore binding affinities of human mAbs to hTie2 at 37° C.Binding at 37° Mab capture format T½ Antibody Analyte ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) (min) H2aM2760N hTie2-His 4.04E+04 3.06E−04 7.60E−09 38hTie2-hFc NT NT NT NT H2aM2761N hTie2-His 4.26E+04 2.87E−04 6.74E−09 40hTie2-hFc NT NT NT NT H1M2055N hTie2-His 1.08E+06 3.36E−04 3.10E−10 34hTie2-hFc NT NT NT NT H1H2304B hTie2-His 3.50E+05 3.67E−03 1.05E−08 3hTie2-mFc 6.60E+05 1.30E−04 1.96E−10 89 H1H2317B hTie2-His 1.74E+053.46E−03 1.98E−08 3 hTie2-mFc 4.60E+05 9.94E−05 2.15E−10 116 H1H2322BhTie2-His 2.12E+05 4.74E−03 2.23E−08 2 hTie2-mFc 5.70E+05 9.72E−051.72E−10 119 H1H2324B hTie2-His 2.78E+05 2.26E−03 8.12E−09 5 hTie2-mFc7.40E+05 1.47E−04 1.99E−10 79 H1H2331B hTie2-His 5.48E+04 4.74E−038.65E−08 2 hTie2-mFc 1.28E+05 1.99E−04 1.56E−09 58 H1H2332B hTie2-His9.58E+04 8.19E−03 8.55E−08 1 hTie2-mFc 1.81E+05 6.52E−04 3.60E−09 18H1H2333S hTie2-His 6.20E+04 9.83E−04 1.58E−08 12 hTie2-mFc NB NB NB NBH1H2337B hTie2-His 8.35E+04 2.41E−03 2.89E−08 5 hTie2-mFc 1.92E+052.55E−04 1.33E−09 45 H1H2338B hTie2-His 5.55E+04 9.93E−04 1.79E−08 12hTie2-mFc NB NB NB NB H1H2339B hTie2-His 6.78E+04 4.88E−03 7.20E−08 2hTie2-mFc 1.93E+05 2.95E−04 1.53E−09 39 H1H2340B hTie2-His 9.75E+049.98E−03 1.02E−07 1 hTie2-mFc 2.00E+05 5.15E−04 2.58E−09 22 H4H2055NhTie2-His 5.49E+05 8.01E−04 1.46E−09 14 hTie2-mFc 1.67E+06 1.55E−049.30E−11 74 Control I hTie2-His 7.48E−04 1.12E−03 1.49E−08 10 hTie2-mFc1.80E+05 1.70E−04 9.40E−10 68 NB = no binding under the conditionstested NT = not tested

TABLE 5 Biacore binding affinities of human mAbs to mTie2 at 25° C.Binding at 25° Mab capture format T½ Antibody Analyte ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) (min) H2aM2760N mTie2-hFc NB NB NB NB H2aM2761N mTie2-hFcNB NB NB NB H1M2055N mTie2-hFc 1.07E+06 1.85E−05 1.73E−11 625 H1H2304BmTie2-hFc NB NB NB NB H1H2317B mTie2-hFc NB NB NB NB H1H2322B mTie2-hFcNB NB NB NB H1H2324B mTie2-hFc NB NB NB NB H1H2331B mTie2-hFc 4.81E+042.72E−03 5.65E−08 4 H1H2332B mTie2-hFc 5.39E+04 5.76E−03 1.07E−07 2H1H2333S mTie2-hFc NB NB NB NB H1H2337B mTie2-hFc 1.87E+05 1.84E−029.83E−08 1 H1H2338B mTie2-hFc NB NB NB NB H1H2339B mTie2-hFc 4.68E+042.63E−03 5.63E−08 4 H1H2340B mTie2-hFc  1.36E_05 6.72E−03 4.96E−08 2H4H2055N mTie2-hFc 1.24E+06 6.19E−06 5.01E−12 1867 Control I mTie2-hFcNB NB NB NB NB = no binding under the conditions tested

TABLE 6 Biacore binding affinities of human mAbs to rTie2 at 25° C.Binding at 25° C. Mab capture format T½ Antibody Analyte ka (Ms⁻¹) kd(s⁻¹) K_(D) (Molar) (min) H2aM2760N rTie2-hFc NB NB NB NB H2aM2761NrTie2-hFc NB NB NB NB H1M2055N rTie2-hFc 9.43E+05 2.55E−05 2.70E−11 454H1H2304B rTie2-hFc NB NB NB NB H1H2317B rTie2-hFc NB NB NB NB H1H2322BrTie2-hFc NB NB NB NB H1H2324B rTie2-hFc NB NB NB NB H1H2331B rTie2-hFcNB NB NB NB H1H2332B rTie2-hFc 1.12E+05 6.18E−03 5.53E−08 2 H1H2333SrTie2-hFc NB NB NB NB H1H2337B rTie2-hFc 1.45E+05 8.46E−03 5.85E−08 1H1H2338B rTie2-hFc NB NB NB NB H1H2339B rTie2-hFc NB NB NB NB H1H2340BrTie2-hFc 1.43E+05 3.71E−03 2.59E−08 3 H4H2055N rTie2-hFc 1.07E+061.00E−06 9.31E−13 11550 Control I rTie2-hFc NB NB NB NB NB = no bindingunder the conditions tested

Example 4. Epitope Mapping of Tie2 mAbs Using Luminex Beads

To determine domain binding for the anti-Tie2 antibodies, several hTie2receptor extracellular domain-deletion constructs were covalently linkedto luminex xMAP beads (10 μg/ml of each protein per 10⁷ beads). Theconstructs are depicted in FIG. 1 and are designated as follows: hTie2(Ig1-Ig2-EGF) (SEQ ID NO:7); hTie2 (Ig2-EGF) (SEQ ID NO:8); hTie2 (EGF)(SEQ ID NO:9); hTie2 (Ig3-FN) (SEQ ID NO:10); and hTie2 (FN) (SEQ IDNO:11). Also tested in this Example were full-length hTie2-mFc (SEQ IDNO:4) and hTie1-hFc (SEQ ID NO:12) ectodomain constructs.

For binding, 25 μl of anti-Tie2 antibody (25 μg/ml) was added to 75 μlof the above created Luminex bead mixture (3×10³ beads per construct) inbinding buffer (PBS, 0.05% Tween 20, 1 mg/ml BSA, 0.05% sodium azide)into a 96 well filter plate (Millipore). Incubation was at roomtemperature (RT) for 90 min or overnight at 4° C. with shaking. Thebeads were washed 3× with washing buffer (PBS+0.05% Tween 20)resuspended in 100 μl binding buffer containing PE(phycoerythrine)-labeled anti human kappa or PE-labeled anti mouse Fabsecondary antibody and incubated at RT for 45 min with shaking. Sampleswere washed 2× more and binding signal (MFI) for each bead wasdetermined using a Luminex L200 or FLEXMAP3D instrument. Bead linkedhuman Tie2 was used as positive control and bead linked human Tie1 wasused to measure family cross-reactivity. In general MFI signals greaterthan 500 units represent significant binding. Results are summarized inTable 7.

TABLE 7 Epitope mapping of Tie2 mAbs hTie2 hTie2 Predicted hTie2- hTie1-hTie2 (Ig1, hTie2 hTie2 (Ig3, FN)- Binding Antibody mFc hFc (EGF) Ig2,EGF) (Ig2, EGF) (FN)-hFc hFc Domain H2aM2760N 1668 15 4 9392 6245 341330 Ig1, Ig2 H2aM2761N 8503 135 10205 11657 8568 3403 14509 EGF, Ig3H1M2055N 753 112 90 2022 873 43 40 Ig1, Ig2 H1H2304B 4250 40 8901 87767648 5 6 EGF repeat H1H2317B 4853 37 9611 8966 8325 9 11 EGF repeatH1H2322B 4040 47 9901 9161 9167 10 13 EGF repeat H1H2324B 4506 31 97739438 8686 7 10 EGF repeat H1H2331B 5766 118 8 6 10 8097 6568 FN repeatH1H2332B 4783 32 3 3 4 6711 6044 FN repeat H1H2333S 1875 10 3 2 2 6 6 NDH1H2337B 5208 48 9 7 9 6425 7368 FN repeat H1H2338B 1385 9 4 5 5 6 8 NDH1H2339B 2913 62 6 5 6 4529 3838 FN repeat H1H2340B 3009 22 4 4 6 51494599 FN repeat Control I 4901 7 3 4 5 6105 4554 FN repeat ND = notdetermined

From the above results, it can be concluded that H2aM2760N and H1M2055Nbind to the Ig1/Ig2 domains of Tie2; H2aM2761N binds to the EGF and Ig3domains of Tie2; H1H2304B, H1H2317B, H1H2322B and H1H2324B bind to theEGF domain of Tie2; and H1H2331B, H1H2332B, H1H2337B, H1H2339B,H1H2340B, and Control I bind to the FN domain of Tie2.

Example 5. Epitope Mapping of H4H2055N Binding to Tie2 by H/D Exchange

Experiments were conducted to more precisely define the amino acidresidues of Tie2 with which H4H2055N interacts. (H4H2055N is a fullyhuman IgG4 version of the antibody H1M2055N produced from hybridomaPTA-12295.) For this purpose H/D exchange epitope mapping was carriedout. A general description of the H/D exchange method is set forth ine.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engenand Smith (2001) Anal. Chem. 73:256A-265A.

To map the binding epitope(s) of antibody H4H2055N on Tie2 via H/Dexchange, a recombinant construct consisting of the Ig1, Ig2 and EGFextracellular domains of human Tie2 (hTie2 (Ig1-Ig2-EGF); SEQ ID NO:7)was used. Antibody H4H2055N was covalently attached toN-hydroxysuccinimide (NHS) agarose beads (GE Lifescience).

In the ‘on-solution/off-beads’ experiment (on-exchange in solutionfollowed by off-exchange on beads), the ligand (hTie2 Ig1-Ig2-EGF) wasdeuterated for 5 min or 10 min in PBS buffer prepared with D₂O, and thenbound to H4H2055N beads through a 2 min incubation. The Tie2-bound beadswere washed with PBS aqueous buffer (prepared with H₂O) and incubatedfor half of the on-exchange time in PBS buffer. After the off-exchange,the bound Tie2 was eluted from beads with an ice-cold low pH TFAsolution. The eluted Tie2 was then digested with immobilized pepsin(Thermo Scientific) for 5 min. The resulting peptides were desaltedusing ZipTip® chromatographic pipette tips and immediately analyzed byUltrafleXtreme matrix assisted laser desorption ionization time offlight (MALDI-TOF) mass spectrometry (MS).

In the ‘on-beads/off-beads’ experiment (on-exchange on beads followed byoff-exchange on beads), Tie2 was first bound to H4H2055N beads and thenincubated for 5 min or 10 min in D₂O for on-exchange. The followingsteps (off-exchange, pepsin digestion, and MS analysis) were carried outas described for the ‘on-solution/off-beads’ procedure. The centroidvalues or average mass-to-charge ratios (m/z) of all the detectedpeptides were calculated and compared between these two sets ofexperiments.

The results are summarized in Table 8 which provides a comparison of thecentroid m/z values for all the detected peptides identified by liquidchromatography-matrix assisted laser desorption ionization (LC-MALDI) MSfollowing the H/D exchange and peptic digest procedure. Likely due todisulfide bonds, the Tie2 sequence coverage detected from a singleMALDI-TOF spectrum is relatively low. Nevertheless, more than half ofthe detected peptic peptides gave similar centroid values for both theon-solution/off-beads and on-beads/off-beads protocols. Three segmentswith corresponding residues 88-106, 139-152, and 166-175 had deltacentroid values 0.20 m/z in both experiments. For purposes of thepresent Example a positive difference (Δ) of at least 0.20 m/z in bothexperiments indicates amino acids protected by antibody binding.Segments meeting this criterion are indicated by bold text and anasterisk (*) in Table 8.

TABLE 8 H4H2055N Binding to Tie2 Experiment I Experiment II Residues 5min on-/2.5 min off-exchange 10 min on-/5 min off-exchange (of SEQ IDOn-solution/ On-Beads/ On-solution/ On-Beads/ NO: 7) Off Beads Off-BeadsΔ Off Beads Off-Beads Δ 20-33 1568.70 1568.63 0.07 1568.63 1568.63 0.0026-33 1036.17 1036.16 0.01 1036.27 1036.21 0.07 38-50 1572.91 1572.93−0.02 1572.78 1572.98 −0.20 39-50 1425.38 1425.39 0.00 1425.54 1425.450.08 42-53 1440.52 1440.44 0.09 1440.62 1440.52 0.10 42-58 2041.432041.42 0.01 2041.58 2041.41 0.17 78-87 1124.17 1124.15 0.03 1124.201124.14 0.07 88-95 1049.49 1049.46 0.02 1049.46 1049.54 −0.08  88-101*1767.78 1767.51 0.27 1767.79 1767.57 0.22  88-102* 1881.08 1880.66 0.421881.14 1880.69 0.45  93-101* 1127.80 1127.57 0.24 1127.80 1127.61 0.19 93-102* 1241.46 1240.83 0.63 1241.34 1240.83 0.51  96-102* 850.54850.22 0.32 850.55 850.25 0.30  96-106* 1233.29 1232.66 0.64 1233.431232.65 0.78  139-151* 1514.22 1513.16 1.06 1514.49 1513.21 1.28 139-152* 1643.17 1642.02 1.16 1643.44 1642.06 1.38 152-165 1498.871498.73 0.14 1498.88 1498.77 0.11 152-166 1662.09 1662.01 0.07 1662.101662.08 0.02 153-165 1369.70 1369.64 0.06 1369.77 1369.66 0.11  166-175*1115.07 1114.59 0.47 1115.04 1114.65 0.39  167-175* 951.73 951.30 0.42951.74 951.33 0.41

Since the peptide fragment (m/z of 1049.4) corresponding to residues88-95 did not show deuteron retention (Δ=0) after off-exchange, thefirst protected segment (88-106) can be reduced to only include residues96-106. Thus the three regions 96-106, 139-152, and 166-175 of SEQ IDNO:7 are protected from full off-exchange by H4H2055N binding to Tie2after on-exchange.

The Ig1 domain of Tie2 consists of amino acids 1-97 of SEQ ID NO:7; theIg2 domain of Tie 2 consists of amino acids 98-186 of SEQ ID NO:7; andthe EGF repeat domain consists of amino acids 187-321 of SEQ ID NO:7.Thus the first binding region (amino acids 96-106 of SEQ ID NO:7)includes the last two amino acids of the Ig1 domain and the first 9amino acids of the Ig2 domain. The second and third binding regions(amino acids 139-152 and 166-175 of SEQ ID NO:7) are located entirelywithin the Ig2 domain.

It should also be noted that the second and third binding regions (aminoacids 139-152 and 166-175 of SEQ ID NO:7) lie within the amino acidregion of Tie 2 that directly contacts its cognate ligand,Angiopoietin-2, based on the co-crystal structure by Barton et al.(2006, Nat Struct Mol Biol 13(6) 524-532). Therefore, this examplesuggests that antibody H4H2055N binds a discontinuous epitope primarilywithin the Ig2 domain of human Tie2 (with one or two potential aminoacid contacts within the C-terminal portion of the Ig1 domain), and thatbinding to these regions correlates with the ability of H4H2055N toblock the interaction between Tie2 and Ang2 as illustrated in Examples 6and 7 herein.

Example 6. Assessment of the Ability of Anti-Tie2 Antibodies to Blockthe Interaction Between Tie2 and Angiopoietins

Tie2 is known to interact with angiopoietins (Ang1, Ang2, Ang3 andAng4). An initial set of experiments was therefore conducted to evaluatethe ability of anti-Tie2 antibodies to block Tie2 binding to Ang2. Inthis first set of experiments, a quantitative blocking immunoassay wasutilized. Briefly, solutions of 0.8 nM biotinylated ecto-Tie2 fused to6×His (SEQ ID NO:2) were premixed with anti-Tie2 antibody ranging from˜50 nM to 0 nM in serial dilutions. After a 1-hour incubation at roomtemperature, the amount of Tie2-His bound to plate coated Bow-Ang2 wasmeasured by sandwich ELISA. Bow-Ang2 (SEQ ID NO:13), an Ang2 constructthat comprises the Fc domain of hIgG1 flanked by one FD domain of Ang2at both termini, was coated at 2 μg/ml on a 96 well microtiter plate andblocked with BSA. Plate-bound biotin-Tie2-His was detected using HRPconjugated streptavidin, and developed using BD OptEIA™ (BD BiosciencesPharmingen, San Diego, Calif.). Signals of OD₄₅₀ nm were recorded anddata was analyzed using GraphPad Prism to calculate IC₅₀ values. Resultsare summarized in Table 9. IC₅₀s were defined as the amount of antibodyrequired to reduce 50% of biotin-Tie2-His detectable to plate boundBow-Ang2 ligand. In Table 9, an antibody is designated an “enhancer” ifthe addition of the antibody increases the amount of Tie2-His bound tothe Bow-Ang2-coated surface relative to the no antibody control.

TABLE 9 IC₅₀ values for anti-Tie2 Mabs blocking of hTie2 to plate boundBow-Ang2 Result in Blocking of binding Blocking to Bow-Ang2 AntibodyELISA IC₅₀ (M) H2aM2760N Enhancer N/A H2aM2761N Enhancer N/A H1M2055NEnhancer N/A H1H2304B Enhancer N/A H1H2317B Enhancer N/A H1H2322BEnhancer N/A H1H2324B Enhancer N/A H1H2331B Enhancer N/A H1H2332BEnhancer N/A H1H2333S Enhancer N/A H1H2337B Enhancer N/A H1H2338BEnhancer N/A H1H2339B Blocker 1.89E−10 H1H2340B Blocker 1.03E−09H4H2055N Blocker 1.06E−09 Control I Enhancer N/A

This first set of experiments demonstrates that anti-Tie2 antibodiesH1H2339B, H1H2340B and H4H2055N are able to block the interactionbetween Ang2 and Tie2 in an ELISA format.

A further set of experiments was conducted to evaluate the ability ofH4H2055N to block Tie2 binding to Ang2 and other members of theangiopoietin family (Ang1, Ang3 and Ang4). In this set of experiments,an Octet Red biosensor was employed using two experimental formats. Inthe first format, hTie2.mFc (SEQ ID NO:4; 10 μg/ml) or a negativecontrol was captured on anti-mFc Octet sensor tips for 5 min. Thecaptured sensor tip surfaces were then saturated with H4H2055N orControl I antibody by dipping into wells containing 300 nM of respectivemonoclonal antibodies for 10 min. Finally the sensor tips were placedinto wells containing 100 nM of hAng1 (R&D Systems, Inc., Minneapolis,Minn., Cat 923-AN/CF; Accession #Q5HYA0), hAng2 (R&D Systems, Inc.,Minneapolis, Minn., Cat 623-AN/CF; Accession #015123), mAng3 (R&DSystems, Inc., Minneapolis, Minn., Cat 738-AN/CF; Accession #Q9WVH6) orhAng4 (R&D Systems, Inc., Minneapolis, Minn., Cat 964-AN/CF; AccessionQ9Y264) for 5 min. Binding response at each step of the experiment wasmonitored and the binding of different angiopoietins to the monoclonalantibody saturated receptor surfaces was plotted (FIG. 2). Asillustrated by FIG. 2, H4H2055N blocked the binding of all fourangiopoietins to the hTie2.mFc surface. The Control I antibody did notblock binding of any of the angiopoietins to the Tie2-coated surfaces.

In the second assay format, hTie2.mFc (10 μg/ml) or negative control wascaptured on anti-mFc coated Octet sensor tips (5 min), followed byplacing the tips into different wells containing 100 nM of angiopoietin(hAng1, hAng2, mAng3 or hAng4) for 10 min. Lastly, the senor tips wereplaced into wells containing 300 nM of H4H2055N or Control I (5 min). Asin format 1, the binding response at each step was monitored and thebinding response was plotted (FIG. 3). This format demonstrated that,unlike Control I, the binding of H4H2055N is diminished by the alreadybound angiopoietins on the Tie2-coated surfaces. Thus, H4H2055N competeswith the angiopoietins (Ang1, Ang2, Ang3 and Ang4) for binding to Tie2.

Example 7. Assessment of the Ability of Anti-Tie2 Antibodies to BlockAngiopoietin-Mediated Tie2 Signaling

To further characterize anti-hTie2 mAbs of the invention, the ability ofthe antibodies to block Tie2-induced luciferase activity was explored.Briefly, HEK293 cells were stably transfected with hTie2, and the top10% of Tie2 expressing cells were isolated via FACS. These cells werethen transduced with a serum response element (SRE)-dependent luciferasereporter lentivirus (SA Biosciences), and a clonal population (293-TIE2/SRE-Luc) with robust response to tetrameric forms of angiopoietin-1(Bow-Ang1, SEQ ID NO:14) and angiopoietin-2 (Bow-Ang2, SEQ ID NO:13) wasisolated.

For assays, 293-Tie2/SRE-Luc cells were seeded (30,000 cells per well)in 96-well plates one day prior to treatment. Dose-response curves weregenerated with serial dilutions of Bow-Ang1 (BA1) or Bow-Ang2 (BA2)incubated with cells for 4 h before measurement of luciferase activity.For Tie2 inhibition, cells were treated with serially diluted Tie2 mAbsin the presence of a 1 nM constant dose of BA1 or BA2 for 4 h.Luciferase activity was measured on a Victor luminometer followingincubation with One-Glo™ (Promega) reagent. Three classes of antibodieswere observed: Blockers, defined as a reduction in Tie2 dependentluciferase activity upon addition of antibody; Activators, defined as anincrease in Tie2 dependent luciferase activity upon addition ofantibody; and Not Active, defined as no significant change in signalupon their addition. For “Not Active” antibodies, no EC₅₀ or 1050 valuewas calculated. Results are summarized in Table 10.

TABLE 10 IC₅₀ values for Anti-Tie2 antibodies EC₅₀ or EC₅₀ or IC₅₀ ± SEMIC₅₀ ± SEM Mab Class (10⁻⁹M) Mab Class (10⁻⁹M) Antibody (BowAng1)BowAng1 (BowAng2) BowAng2 H2aM2760N Blocker 11.6 ± .81 Blocker 2.8 ± .14H2aM2761N Blocker, not  5.2 ± 1.3 Blocker, not to 1.1 ± .11 to baselinebaseline H4H2055N Blocker 0.69 ± .16 Blocker 0.23 ± .03  H1H2304B Noactivity NA No activity NA H1H2317B No activity NA No activity NAH1H2322B No activity NA No activity NA H1H2324B No activity NA Noactivity NA H1H2331B Activator 13.6 ± 3.1 Activator 5.1 ± 1.9 H1H2332BActivator  2.5 ± 1.2 Activator 3.7 ± .15 H1H2333B No activity NA Noactivity NA H1H2337B Activator  1.1 ± .02 Activator 4.7 ± .80 H1H2338BNo activity NA No activity NA H1H2339B Activator 10.3 ± .23 Activator13.0 ± 3.5  HIH2340B Activator 1.41 ± .01 Activator 2.1 ± .04 Control IActivator 0.76 ± .50 Activator 1.4 ± .03 IC₅₀ values are reported forBlockers, and EC₅₀ values are reported for Activators

Anti-Tie2 antibodies H2aM2760N, H2aM2761N and H4H2055N were identifiedas blockers in this experimental system. Anti-Tie2 antibodies H1H2332B,H1H2339B and H1H2340B were identified as activators of both BowAng1 andBowAng2.

Example 8. Assessment of the Anti-Tumor Activity of an Anti-Tie2Antibody In Vivo

An exemplary anti-Tie2 antibody (H2M2055N) was tested for its ability toinhibit the growth of human tumor xenografts in immunocompromised mice.Briefly, either human colorectal HT29 or Colo205 tumors were grown inSCID mice. When tumors were palpable (100 mm³ for HT29 and 400 mm³ forColo205) animals were treated biweekly with Fc protein (10 mg/kg) oranti-Tie2 antibody (H1M2055N; 10 mg/kg). At the end of treatment (31days for HT29 & 14 days for Colo205), the percent tumor growthinhibition (% TGI) was determined (Table 9) and tumor tissue washarvested and utilized for vessel density analysis. Vascular density wasassessed in 30 μm thick OCT tumor sections by CD31 immunohistochemistry.NIH Image software was utilized to determine the % area vessel densityin the tumor tissue sections. Results are illustrated graphically inFIGS. 4-7.

TABLE 11 Percent Decrease in Tumor Growth with Anti-Tie2 Mab TreatmentHT29 Colo205 Avg Tumor % Avg Tumor % Growth (mm3) Decrease Growth (mm3)Decrease from start of in Tumor from start of in Tumor Antibodytreatment Growth treatment Growth Fc protein 100 400 (10 mg/kg) H2M2055N100 27.5 400 77 (10 mg/kg)

As shown in this Example, the anti-Tie2 Ab, H1M2055N, not only decreasedtumor growth (Table 9), but also significantly decreased vessel densityin both HT29 (25%, FIG. 5) and Colo205 (40%, FIG. 7) xenograft models.

Example 9: Tie2 Activating mAbs for Vascular Leak

HUVEC cells were seeded on collagen-coated 24-well transwell inserts (1μm filter) and grown for 48 h to confluence. Transepithelial electricalresistance (TEER) was measured on a daily basis to confirm monolayerformation (EVOM-2, World Precision Instruments). Cells were treated for24 h with TNF-α in the presence or absence of combinations of Tie2activating antibodies (either H4H2340P/H4H2339P or H4H2332P/H4H2339P).Permeability was determined by measurement of the fluorometric signal(485/535 nm) in the abluminal chamber 1 hour after addition of 0.5 mg/mL(75 μg/insert) FITC-Dextran (70 kD) to the luminal chamber, using theSpectramax i3x Detection Platform (Molecular Devices). Percentpermeability relative to the untreated control was calculated using thefollowing equation: %permeability=(Fluorescence_(sample)−Fluorescence_(Min Signal Ctrl (Untreated)))/(Fluorescence_(Max Signal Ctrl (No cells))−Fluorescence_(Min Signal Ctrl (Untreated)))).

As shown in FIG. 8 and Table 12, below, on confluent HUVECs, theaddition of TNF-α leads to an increase in permeability compared tountreated HUVECs, suggesting a disruption of cell to cell contact.Addition of 166.7 nM of either combination of Tie2 activating antibodies(H4H2340P/H4H2339P or H4H2332P/H4H2339P) reduced the passage ofFITC-Dextran through the disrupted HUVEC monolayer by 30.7% and 53.8%,respectively compared to TNF alone suggesting a decrease in theformation of intercellular gaps in the endothelial barrier.

TABLE 12 Average percent permeability relative to the untreated cellcontrol. TNF-α + REGN1945 (hIgG4 Ctrl) Untreated TNF-α + TNF-α +H4H2340P/ Isotype No cells cells TNF-α H4H2332P/H4H2339P H4H2339PControl 100.00 ± 7.14 0.00 ± 0.15 10.81 ± 0.88 5.00 ± 0.45 7.50 ± 0.389.60 ± 0.63

Example 10: Tie2 Activating mAbs for Vascular Leak in an In Vivo MouseModel of Influenza and Sepsis

Seven-week old, female BALB/c mice were anesthetized with aketamine/xylazine mixture before receiving an intranasal infection with10× mouse LD₅₀ (450 PFUs/mouse) of A/Puerto Rico/08/1934 (H1N1) in 20 μLsaline. AT 2- and 4-days post-infection (p.i.) the mice were treatedwith a subcutaneous dose of 20 mg/kg of each Tie2 mAb combinations (40mg/kg total of H4H2340P/H4H2339P or H4H2332P/H4H2339P) and/or isotypecontrol. On day 3 p.i., mice received an intravenous dose of 12 mg/kg ofthe anti-influenza A group 1 mAb H1H11729P, an anti-influenza HA orisotype control (see, e.g., US 2016/0176953, incorporated by reference).All mice were monitored daily for 14 days. Death was determined by a 25%body weight loss threshold that was authorized by the RegeneronInstitutional Animal Care and Use Committee. Results are reported inTable 13 as percent survival.

TABLE 13 Therapeutic administration of activating Tie2 mAbs incombination with anti-influenza A hemagglutinin mAb Numbersurviving/total N Group Treatment (percent) 1 Uninfected saline control 5/5 (100) 2 H1H11729P + H4H2340P/H4H2339P 4/5 (80) 3 H1H11729P +H4H2332P/H4H2339P 3/5 (60) 4 H1H11729P + IgG4 isotype control 2/5 (40) 5H4H2340P/H4H2339P + IgG1 isotype control 1/5 (20) 6 H4H2332P/H4H2339P +IgG1 isotype control 1/5 (20) 7 IgG1 isotype control + IgG4 isotypecontrol 0/5 (0) 

As shown in Table 13, the Tie2 mAbs H4H2340P/H4H2339P orH4H2332P/H4H2339P showed robust efficacy in combination with theanti-influenza A group 1 hemaglutinning mAb, H1H17729P in treating miceinfected with a historical influenza strain as shown in Table 14 andFIG. 9. The combination of a single dose of H1H11729P with two doses ofH4H2340P/H4H2339P resulted in 80% survival compared to either treatmentsgiven alone with isotype control mAbs (40% and 20%, respectively; Table13 and FIGS. 9 and 10). The combination of a single dose of H1H11729Pwith two doses of H4H2332P/H4H2339P resulted in 60% survival compared to40% and 20% survival with either treatment alone. Mice dosed withisotype control mAbs only all died by day 7 post-infection. Theseresults show that therapeutic administration of activating Tie2 mAbs incombination with an anti-pathogen-specific mAb improves survivalcompared to treatment with the anti-hemagglutinin-specific mAb aloneafter lethal challenge with Influenza A.

Ten-week old, male C57/BL6 mice were treated with a subcutaneous dose of20 mg/kg of each Tie2 mAb combination (40 mg/kg total ofH4H2340P/H4H2339P or H4H2332P/H4H2339P) or isotype control 48 h beforeintoxication. On the day of intoxication (time 0), LPS was reconstitutedto a dose of 25 mg/kg mouse by sonication and LPS was administeredintraperitonally. All mice were monitored daily for 7 days every 6hours. Death was determined by a 20% body weight loss threshold. Resultsare reported as percent survival.

The Tie2 mAbs H4H2340/H4H2339P showed efficacy when givenprophylactically in an LPS sepsis model (Table 14 and FIG. 10). Micethat received 40 mg/kg of the H4H2332P/H4H2339P combination, isotypecontrol or PGS only were all dead by 24 or 48 hours post-intoxication.Mice that received 40 mg/kg total of the H4H2340P/H4H2339P combination48 h before intoxication showed delayed death and 20% survival. Theseresults show that prophylactic administration of a combination ofactivating Tie2 mAbs improves survival and delays death in a very lethalmodel of sepsis.

TABLE 14 Prophylactic administration of activating Tie2 mAbsH4H2340P/H4H2339P delays death and improves survival in a lethal modelof E. coli 0111:B4 LPS intoxication over isotype/untreated controls.Number surviving/total Group Treatment N (percent) 1 Unintoxicatedsaline control  5/5 (100) 2 H4H2340P/H4H2339P  1/5 (20) 3H4H2332P/H4H2339P 0/5 (0) 4 IgG4 isotype control 0/5 (0) 5 Saline alone0/5 (0)

As shown in FIG. 10, prophylactic administration of activating Tie2mAbs, H4H2340P/H4H2339P resulted in a delay in death and increase insurvival after intoxication. Mice received a single dose ofH4H2340P/H4H2339 or H4H2332P/H4H2339P (40 mg/kg total on 2 days beforeintoxication, SC). LPS was administered IP at time 0. Results from oneexperiment (N=5) are shown.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method for inhibiting an influenza infection ina patient, the method comprising administering to the patient a humanantibody or antigen-binding fragment thereof that specifically bindshuman Tie2 in the fibronectin (FN) repeat domain and activatesangiopoietin-mediated Tie2 signaling in vitro.
 2. The method of claim 1,wherein the antibody or antigen-binding fragment is an antibody with ahuman IgG4 heavy chain constant region.
 3. The method of claim 1,wherein the antibody or antigen-binding fragment that specifically bindshuman Tie2 is administered with an anti-influenza HA antibody.
 4. Themethod of claim 1, wherein the antibody or antigen-binding fragment thatspecifically binds human Tie2 is administered with an antibiotic, anantiviral agent, an analgesic, a non-steroidal anti-inflammatory drug(NSAID) or a corticosteroid.